One of the major goals of current functional genomics research is to establish correlations between gene expression levels and particular cellular states of interest (e.g., disease states, certain developmental stages, states associated with exposure to particular environmental stimuli and states resulting from administration of particular therapeutic treatments). The establishment of such correlations has the potential to provide significant insight into the mechanism of disease, cellular development and differentiation, as well as being of value in the identification of new therapeutics, drug targets and/or disease markers.
Historically, functional genomic studies have focused on mRNA levels in making such correlations. This focus is due in large part because of the generic nature of the methodology for detecting different mRNAs, namely the detection of hybridization between nucleic acid probes and target mRNA molecules. Recent research, however, indicates that often mRNA expression does not correlate well with protein expression, and even less well with protein accumulation or content. Such results are not particularly surprising since many factors affect protein levels independent of transcriptional control, including for example, differences in translational efficiency, turnover rates, whether the protein is compartmentalized or expressed extracellularly, and post-translational modifications. Thus, profiling proteins rather than mRNA is often the preferred approach for conducting functional genomic studies. This is particularly true since proteins are the cellular agents responsible for the catalytic activity of a cell or tissue; hence, by monitoring protein expression, one is able to more directly monitor the actual agents responsible for the biological processes that occur within the cell or tissue.
Various techniques have been utilized in analyzing the protein content of a cell or tissue. Two-dimensional (2-D) gel electrophoresis is one of the more widely utilized techniques for performing such analyses. As the name implies, the method involves separating proteins within a cell or tissue into two dimensions on an electrophoretic separation matrix. The separated proteins are then typically detected by various staining protocols thus yielding a multitude of spots on the gel. If the separation is done under appropriate conditions, the location of the proteins can be used to identify particular proteins, or at least to provide a “fingerprint” of the proteins present in particular cells. There has been a proliferation of protein gel image databases to assist in the identification and comparison of protein levels in different cells and tissues. An example of such a database is the Protein-Disease Database maintained by the National Institutes of Health (NIH). A significant limitation of such methods, however, is the difficulty in identifying the proteins present at each of the spots on a gel.
Phage-display technology is a technology that has been widely utilized in protein analysis. However, this technology has been utilized primarily to produce-and screen large libraries of polypeptides to identify polypeptides capable of specifically binding to particular targets (see, e.g., Cwirla et al., Proc. Natl. Acad. Sci. USA 87:6378–6382 (1990); Devlin et al., Science 249:404–406 (1990); Scott and Smith, Science 249:386–388 (1990); and Ladner et al., U.S. Pat. No. 5,571,698). Phage display methods typically involve the insertion of random oligonucleotides into a phage genome such that they direct a bacterial host to express peptide libraries fused to phage coat proteins (e.g., filamentous phage pIII, pVI or pVIII). Libraries of up to 1010 individual members can be routinely prepared in this way. Incorporation of the fusion proteins into the mature phage coat results in the peptide encoded by the heterologous sequence being displayed on the exterior surface of the phage, while the heterologous sequence encoding the peptide resides within the phage particle.
The utility of this technology lies in the physical association between the displayed peptide and the genetic material encoding it; this association permits the simultaneous mass screening of very large numbers of phage bearing different peptides. Phage displaying peptides having binding specificity for a particular target can be enriched by affinity screening against the target. The identity of such peptides can be determined from the heterologous sequence contained in the phage displaying the peptide.
Display technology can be utilized to prepare recombinant antibody display libraries for use in the analysis of protein samples. Often such libraries are produced as phage display libraries. Conducting analyses with such libraries is complicated by the fact that in such libraries it is the displayed antibody, rather than the target protein specifically bound by the antibody, that is encoded by the heterologous nucleic acid sequence within the display package (typically a bacteriophage).
Hence, although various methods for conducting certain types of protein analysis have been developed, a significant impediment to analyzing protein expression as a means to gain insight into biological processes is the lack of a generic detection reagent and methodology that is comparable to the ability to use nucleic acid probes in hybridization reactions as detection reagents to detect the presence of complementary nucleic acids.